Non-corrosive sterilant composition

ABSTRACT

A non-corrosive, liquid, aqueous sterilant composition (as a concentrate or ready-to-use solution), which may be provided in two parts which are mixed prior to application, may comprise a peracid (in an equilibrium solution with an underlying carboxylic acid or mixtures of alkyl carboxylic acids and peroxide), inorganic buffering agent, and water. It has been found that the use of this simplified system, even in the absence of additional components which have been thought to be desirable for sterilants used on metal parts (e.g., copper and brass corrosion inhibitors, chelating agents, anti-corrosive agents) display excellent performance and that these additional components are not necessary, and that the presence of these additional materials at least complicates disposal of the spent solutions and could complicate compatibility of the sterilant solutions with some polymeric materials, especially where organic materials are used as the additional components, which organic materials may interact with, dissolve or solubilize in the polymeric materials.

This application claims the benefit of U.S. Provisional application No.60/109,565, filed Nov. 23, 1998.

NON-CORROSIVE STERILANT COMPOSITION

The present invention relates to compositions which can be used tosafely and effectively disinfect surfaces and articles againstmicrobiological forms. The compositions are easily handled, tend to benon-corrosive to the types of polymeric, elastomeric and metal surfacesfound in medical instruments, are relatively shelf-stable, and may beprepared quickly and easily by simply blending component solutions.

The importance of the sterilization of medical instruments and implantshas been understood for more than two centuries. The need forsterilization has become even more important recently with theappearance of strains of microbiological forms which are resistant toconventional microbiocides such as antibiotics. It has become veryimportant to sterilize medical devices to kill or remove the moreresistant strains of microbiological forms before they infect a patient.Additionally, the sterilants must be generally effective againstmicroorganisms covering a wide range of classes and species, with U.S.Government standards requiring efficacy against both bacteria andspores.

Sterilization of medical devices has been performed for many years byimmersing the medical devices in an atmosphere which is antagonistic tothe survival of the microbiological forms. Among the environments whichhave been used to attempt to sterilize medical instruments include, butis not limited to, steam, alcohols, ethylene oxide, formaldehyde,gluteraldehyde, hydrogen peroxide, and peracids. Each of these materialshas its benefits and limitations. Ethylene oxide tends to be veryeffective against a wide range of microorganisms, but it is highlyflammable and is generally used in a gas phase which may require morestringent environmental restraints than would a liquid. Alcohols aresimilarly flammable and must be used in very high concentrations. Steamhas a more limited utility, having to be used in a controlled andenclosed environment, requiring the use of large amounts of energy tovaporize the water, and requiring prolonged exposure periods to assureextended high temperature contact of the steam with the organisms.Hydrogen peroxide has limited applicability because it is unstable andnot as strong as some other sterilants. The peracids have become morefavorably looked upon, but they tend to be corrosive (being an oxidizingacid) and are not shelf stable.

U.S. Pat. No. 5,508,046 describes a stable, anticorrosive peraceticacid/peroxide sterilant comprising a concentrate including peraceticacid, acetic acid, hydrogen peroxide (in a ratio of 1:1 to 11:1 totalacid/hydroxide), and 0.001 to 200 parts per million of stabilizers suchas phosphonic acids and sodium pyrophosphates. The concentrates arediluted about 20 to 40 times so that the maximum concentration ofstabilizer in the use solution would be about 10 parts per million. Thestabilizers are described as acting as chelating agents by removingtrace metals which accelerate the decomposition of the peroxides.

U.S. Pat. No. 5,616,616 describes a room temperature sterilantparticularly useful with hard tap water comprising an ester of formicacid, an oxidizer (such as hydrogen peroxide or urea hydrogen peroxide),perfonuic acid and water. The use of corrosion inhibitors (such asbenzotriazoles, azimidobenzene, and benzene amide) and stabilizers(unnamed) is also generally suggested.

U.S. Pat. No. 5,077,008 describes a method of removing microbialcontamination and a solution for use with that method. The solutioncomprises a combination of five ingredients in water: 1) a strongoxidant (including, for example, organic peroxides, peracids, anchloride releasing compounds, with peracetic acid in a concentration of0.005 to 1.0% being preferred), 2) a copper and brass corrosioninhibitor (e.g., triazoles, azoles and benzoates), 3) a buffering agent(including, for example, phosphate), 4) at least one anti-corrosiveagent which inhibits corrosion in at least aluminum, carbon steel andstainless steel selected from the group consisting of chromates anddichromates, borates, phosphates, molybdates, vanadates and tungstates,and 5) a wetting agent. A sequestering agent may be used to prevent thephosphates from causing precipitation in hard water.

U.S. Pat. Nos. 4,892,706 and 4,731,22 describe automated liquidsterilization systems having a plurality of modules which store thesterilant solution and the rinse solution. U.S. Pat. No. 5.037,623describes a sterilant concentrate injection system which is a spillresistant, vented ampule system for use with sterilization systems.

Medical devices now include many polymeric components for reasons ofmaterial costs and ease of manufacture. Many of the systems andsolutions designed for the sterilization of metal medical devices arenot necessarily suitable for use with polymeric components, and maycause corrosion of the polymeric materials. It is therefore necessary toformulate sterilization compositions which are compatible with bothmetal and polymeric components of the medical devices. It is also alwaysdesirable to provide sterilization systems with fewer components in thecomposition, where the sterilization solutions do not significantlysacrifice microbiocidal activity and do not corrode the materials usedin medical devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the reduction of B. cereus spores at 40° C.

FIG. 2 is a graph showing the reduction of B. cereus spores at 60° C.

FIG. 3 is a graph showing the reduction of B. cereus spores at 40° C.

SUMMARY OF THE INVENTION

A non-corrosive, liquid, aqueous sterilant composition (as a concentrateor ready-to-use solution), which may be provided in two parts which aremixed prior to application, may comprise a peracid (in an equilibriumsolution with an underlying carboxylic acid or mixtures of alkylcarboxylic acids and peroxide), inorganic buffering agent, and water. Ithas been found that the use of this simplified system provides excellentsterilization ability, even in the absence of additional componentswhich have been thought to be desirable for sterilants used on metalparts (e.g., copper and brass corrosion inhibitors, chelating agents,anti-corrosive agents) which have been found to not be necessary. Thepresence of these additional materials at least complicates disposal ofthe spent solutions and could complicate compatibility of the sterilantsolutions with some polymeric materials, especially where organicmaterials are used as the additional components, which organic materialsmay interact with, dissolve or solubilize in the polymeric materials.

The concentration of the components has shown itself to be important inproviding non-corrosive effects towards a wide variety of structuralmaterials in medical devices and yet providing effective sterilizationeffects against spores and bacteria, including tuberculosis bacteria inan acceptable amount of time.

An aqueous sterilant use solution according to the present invention maycomprise a solution having a pH of from 5.0 to 7.0 comprising from 100to 10,000 parts per million of a peroxy acid and 30 to 5000 parts permillion of buffering agent, preferably without any organic anticorrosiveagents. The aqueous sterilant solution may, for example, comprise from100 to 10,000 parts per million of a peroxy acid, 30 to 5000 parts permillion of buffering agent and a catalytically effective amount of acatalyst for peroxygenation of a carboxylic acid by hydrogen peroxide.

The aqueous sterilant solution may consist essentially of a solutionhaving a pH of from 5.0 to 7.0 comprising from 100 to 10,000 parts permillion of a peroxy acid, 30 to 5000 parts per million of bufferingagent and a catalytically effective amount of a catalyst forperoxygenation of a carboxylic acid by hydrogen peroxide.

The method may particularly comprise mixing a first and a secondsolution to form a sterilizing solution comprising a peroxy acid, saidfirst solution comprising a carboxylic acid, hydrogen peroxide andwater, and said second solution comprising a buffering agent for pHbetween about 5 and 7, said sterilizing solution comprising at least 100parts per million of peroxy acid at a pH of 5 to 7, immersing saidarticle in said sterilizing solution for at least 5 minutes to sterilizesaid article, said first solution and second solution being free oforganic anti-corrosion agents for brass and/or copper, and said articlecomprising a medical article having parts made of at least two materialsselected from the group consisting of metals, polymers and rubbers.

DETAILED DESCRIPTION OF THE INVENTION

The aqueous sterilant compositions of the present invention comprise aperacid, water-soluble peroxide source, and carboxylic acid in abuffered solution at pH levels between about 5.0 and 7.0. The use of aninorganic buffering agent also enables the use of slightlywater-soluble, higher molecular weight carboxylic acids in the formationof peroxy acids with the peroxide source thereby reducing the amount ofdeposits from fatty acid residue in the solution. Phosphate buffers areeffective dispersants and suspending agents for these fatty acidresidues.

The peroxy acid useful in the practice of the present invention maycomprise any organic peroxy acid. These acids are well known in the artto be formed from any carboxylic acid containing compound. Normally theyare prepared from carboxylic acids of the formula:

CH₃—(CH₂)n—COOH

wherein n is 0 to 18, preferably 0 to 12 and more preferably 0 to 10,with the corresponding peroxy acid having the formula:

CH₃—(CH₂)n—CO₃H

wherein n is as defined above. The alkyl moiety on the acid, CH₃—(CH₂)n—may be replaced with hydrogen or any, preferably low molecular weight,organic group so that the acid and the resulting peroxy acid may berepresented by: R—CO₂H and R—CO₃H, respectively. The molecular weight ofR could be 1, but preferably'should be between 15 and 155.

Carboxylic acids which are generally useful in the invenetion are thosewhich comprise percarboxylic acids. Percarboxylic acids generally havethe formula R(Co₃H_(n)), where R is an alkyl, arylaklyl, cycloalkyl,aromatic or heterocyclic group, and N is 1, 2, or 3 and named byprefixing the parent acid with peroxy.

The peracid normally exists in an equilibrium state with the original orfundamental acid and the peroxide source, usually hydrogen peroxide.Typical peracids include peracids of C₁ to C₁₂ carboxylic acids such asformic acid, acetic acid, propanoic acid, butanoic acid, pentanoic acid,hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, and the like. The termcarboxylic acids as used in the practice of the present invention,unless otherwise limited, also includes mono- and di-hydroxycarboxylicacids such as glycolic acid, lactic acid and citric acid. An example ofdi-hydroxycarboxylic acid or di-hydroxy is tartaric acid, and alsofumaric acid, which is an unsaturated di-hydroxycarboxylic acid. Diacidssuch as alpha-omega-dicarboxylicpropanoic acid, succinic acid, glutaricacid, adipic acid, and the like may also be used to form di-peracids.Peroxycarboxylic acids may also be present and included within thesolutions of the present invention. Mixtures and combinations of theperacids may also be used in the systems of the invention, as well asother addenda as generally described herein.

The peroxide source is preferably an aqueous solution of hydrogenperoxide, but may also include such alternative peroxide sources assolutions of sodium peroxide, calcium peroxide, alkali salts ofpercarbonate and persulfate, and even organic peroxides such as dicumylperoxide, dialkyl peroxides, urea peroxide, and the like, forming thebasis of the solution of the hydrogen peroxide. The inorganic peroxidesare preferred as the source of the solution of the hydrogen peroxide.The ratio of the peroxy acid to the hydrogen peroxide can alsosignificantly influence the efficacy of the solutions of the invention,with higher ratios of the peroxy acid to the hydrogen peroxidepreferred. For example, its is more desirable to have a ratio of atleast 2:1 or 3:1 (peroxy acid to hydrogen peroxide), and more desirableto have higher ratios of at least 4:1, at least 5:1 or at least 8:1 ormore (peroxy acid to hydrogen peroxide).

The buffering agent is a compound, again preferably an inorganiccompound which will maintain a buffered pH level in the solution of thecomposition between 5.0 and 7.0. Buffering agents include, but are notlimited to phosphates, borates, lactates, acetates, citrates, vanadates,tungstates, and combinations thereof, particularly alkali metal oralkaline metal salts of these agents. The use of phosphates exclusivelyor at least primarily (e.g., at least 50%, at least 65%, at least 75%,or at least 90 or 95% by weight of the buffering agents) is particularlyuseful. Trisodium phosphate has been found to be particularly desirablebecause of its ability to maintain the acid residues of the peroxy acidsin solution where they will not form film in the solution which can bepicked up by any sterilization apparatus or medical device which isbeing sterilized. It is interesting to note that phosphates have beengenerally taught to be avoided in sterilization solutions where hardwater may be contacted because of the potential for calciumprecipitation, yet in the present invention, the presence of phosphatesreduces the formation of organic residue film on the surface of thesolution. The buffering agent alone, even when a phosphate or especiallywhen a phosphate and particularly trisodium phosphate, has been found toreduce corrosion by the solution on all surfaces. The use ofphosphate(s) alone, in the absence of copper and brass corrosioninhibitors has been found to be an effective sterilant, and providenon-corrosive activity against a wide range of structural materials,including, but not limited to rubbers, plastics and metals, such asstainless steel, aluminum, polypropylene, teflon,acrylonitrile/styrene/butadiene, polyolefins, vinyl resins (e.g.,polyvinyl chloride, polyvinylbutyral), silicone resins and rubbers, andpolyurethanes, and provide second tier protection for brass and copper.Although the peracids work more efficiently in their microbiocidalactivity at highly acidic pH levels (below 4.0), those acidic levels aremuch more corrosive. The use of a buffering system which maintains thepH above 5.0 and preferably between about 5.0 and 7.0 still provides amicrobiocidal activity at levels which meet all international standards,using anywhere from 150 to 10,000 parts per million peracid.

The sterilant can be used as a manual system or be used in an automatedsystem. The sterilant can be provided as a one-part or preferably twopart concentrate, with the peracid in one solution and the buffer in thesecond solution. For example, in a two-part system, a peracidconcentrate may be formed having 0.01% to 1% by weight peracid (e.g.,peracetic acid), 0.003% to 1% by weight ppm hydrogen peroxide, 0.01% to1% by weight acid (e.g., acetic acid), and the buffer solution maycomprise, for example, from 0.5 to 75,000 ppm buffering agent (e.g.,anhydrous trisodium phosphate) in water. Mixtures of these types ofaddenda, including the buffering agents and peracids, are clearly usefulin the practice of the present invention. It is preferred that theconcentrates have active ingredient contents at the higher levels ofthese ranges such as 0.1% to 15% by weight peracid, 5% to 80% by weightperoxide, 5% to 80% by weight acid and 0.1% to 15% by weight bufferingagents. The diluted to use solution would preferably contain sufficientactives to provide 0.01% to 1.0% by weight peracid at a pH between about5.0 and 7.0. The use solution need not contain any effective amount ofmany of the additives which prior art systems have required fornon-corrosive effects (such as the organic anti-corrosive agents such asthe triazines, benzotriazoles, azoles and benzoates), and yet provide awider disclosed range of non-corrosivity against the many availablesurfaces of medical devices. The use solutions of the present inventionmay comprise a simplest solution comprising peracid (along with the acidand peroxide in equilibrium), buffering agent in an amount to provide apH of from about 5.0 to 7.0, and water (preferably deionized water).This solution may be modified by the addition of individual agents suchas chelating agents, surfactants (also referred to in the literature forsterilant compositions as wetting agents), and anti-corrosion agents. Atypical concentrate solution which may be diluted to a use solutionmight comprise, 0.1% to 15% by weight peracid, 0.1% to 15% by weightbuffering agent[, with the remainder as water and other addenda asgenerally described herein (e.g., from 99.6 to 78% by weight water).These and other aspects of the invention will be further described byreference to the following, non-limiting examples.

These data show that a preferred range for the concentration of peroxidein the solution (particularly as evidenced by hydrogen peroxide) lessthan 150 ppm, preferably less than 100 up to 80,000 ppm, still morepreferably less than 100, less than 75 and less than 50 ppm. In theexamples, POAA represents peroxyacetic acid, AA represents acetic acid,POOA represents peroxyoctanoic acid, and Oct. Acid represents octanoicacid. Dequest™ are commercially available materials which may be used inthe solutions of the present invention. Dequest™ 2000 comprisesaminotri(methylene-phosphonic acid), Dequest™ 2010 comprises1-hydroxyethylidene-1,1-diphosphonic acid, and Dequest™ 2006 comprisesaminotri(methylene-phosphonic acid)pentasodium salt. Dequest acts as achelator for heavy metals. The data also shows that sporicidal activityof compositions with higher molecular weight peracids increase withhigher proportions of the peracid as compared to the acid.

The presence of a catalyst for the formation of the peracid in thesterilization compositions of the present invention also is a novelaspect of the present invention which could act to maintain the level ofperacid in the solution during use.

Corrosion Example I Experimental

In the following comparison example, a formulation according to thepresent invention comprising 2.69 weight percent of a 13% solution ofperacetic acid made by combining 78% glacial acetic acid, 21% hydrogenperoxide (35% by weight in water), and 1% hydroxyethylenediaminephosphonate was compared to a commercial sterilization formulation (CSF)comprising a mixture of sodium perborate and tetraacetyl ethylenediaminewith a buffer to provide a use solution of pH 8, with its necessarysterilization activator. The CSF composition (referred to as Powder PAA)comprises a powder source of peracetic acid (with a solid peroxidesource) without a buffering agent, and was compared to a liquid solutionof peracetic acid (PAA) made according to the present invention(referred to as Liquid PAA) by admixture of acetic acid and hydrogenperoxide solution with 1% by weight of hydroxyethylenediaminephosphonate catalyst to form the solution of peracetic acid (with theequilibrium amounts of acetic acid and hydrogen peroxide) at a pH of 6.0provided by 3.0% by weight trisodium phosphate. This commercial CSFproduct requires mixing of a dry powder, with a delay required for theactivator TAED (tetra acetyl ethylene diamine) by reaction with sodiumperborate to generate peracetic acid and microbiocidal activity in thecomponents.

Test Parameters

The test was performed on pieces of an Olympus flexible endoscopes usinga washer/disinfector to reduce manual variables. The test parameterswere room temperature conditions, with the following immersion times:

Sample Cycles Immersion Time Liquid PAA 1 10 minutes Powder PAA 1 15minutes Sample Application Time Liquid PAA 24 hours Powder PAA 8 hours

The test was performed by completely immersing separate test pieces S1to S7 and W1 to W28 in each of the solutions.

Test Pieces

Item Parts S1-S7 Parts of endoscope S8 and S9 Insertion tube S10 Lightguide tube W1-W28 Parats of washer/disinfector

Sample Surface No. Material (base) Control Place of the Parts S1A5056BD-H32 Resin black connector to LS painting S2 Polysulfone blackmain body painting S3 SUS304 Resin El. black outside (hidden) coating S4Silicone Rubber — outside S5 Polybutadiene PB-60 — outside S6 Mod. PPOblack main body Polyphenyleneoxide painting S7 A5056BD-H32 Resin blackeyepiece alumite S Polyurethane primary insertion tube coat Z SPolyurethane primary insertion tube coat V S Polyurethane light guidecable W1 Stainless Steel inner pipe system W2 Stainless Steel inner pipesystem W3 epoxy resin + coating heating panel W4 Polyethylene basin W5Polypropylene basin W6 Polyacetate connector W7 Polysulfone part of topcover W8 Silicone Rubber sealing W9 Polyvinyl chloride inner pipe systemW10 Polyvinyl chloride (hard) inner pipe system W11 Acrylic polymerparts in the basin W12 Ethylene/propylene inner pipe system W13Ethylene/propylene rubber inner pipe system W14 Acrylate modified topcover PolyVinylChloride W15 Butyl-nitrile rubber + parts in the basinPhenol W16 Teflon name plate in basin W17 Butyl-nitrile rubber sealingW18 Polyurethane ? W19 Acrylonitrile/butadiene/ top cover styrene W20modified PPO top cover W21 Butyl rubber sealing W22 fluorinated rubbersealing W23 alumina ceramic parts of pump system W24 Teflon parts ofpump system W24 Teflon rubber parts of pump system

Conclusion

The samples were carefully inspected to evaluate the cosmetic effects(corrosion effects) on the various pieces. The first examination(Item 1) was for parts of the endoscope. The second examination (Item 2)was for the insertion tube. The third examination (Item 3) was for thelight guide tube. The fourth examination (Item 4) was for thewasher/disinfector. The samples performed substantially identically,with both solutions showing only a slight cosmetic change in paintedblack surface of the endoscope (S3 surface). No functional or cosmeticchanges were noted on any other sample. The simplicity of use for theLiquid PAA system was very noteworthy, with no delay in mixing orreaction time. The solutions could be directly added into an automatedsystem while the CSF Powder PAA system would have required premixing andactivation time before it could have been used in an automatic system.

Corrosion Example II Experimental

A corrosion study was performed to evaluate peracid containing formulaswith and without buffer addition upon selected metals, plastics andrubbers.

Testing was conducted with two peracid formulation of 500 ppm (parts permillion) peracetic acid (A) and 5000 ppm peracetic acid (B)concentration with buffer; and, two identical formulas (C and Drespectively) with exception of no buffer addtion admixture.

Coupons were completely immersed in 200 mls of defined test solutioncontained in covered 8 ounce glass jars maintained at 50° C. within anenvironmental chamber. Solutions were changed daily. Study was conductedover a 14 day time period. For each test material, a control was alsorun which is a coupon of stated material placed within a covered 8 ounceglass jar having no test solution.

Coupons were pretreated before the corrosion study began, and postreatedbefore final comparitive measurements and visual observations wereperformed. Metal coupons were precleaned according to ASTM Vol. 3.02,G31-72 and 3.02, G1-90 protocol and post-treated accordingly prior tofinal measurement. Test conditions were modified from the ASTM protocolas explained in above paragraph. Plastic and rubber coupons were onlyrinsed with deionized water and air dried prior to corrosion study; and,similarly treated prior to final measurement and visual observation.

Conclusion

Addition of buffer admixture to peracetic acid composition testsolutions significantly improves metals protection. The effect is lessnoticeable on test plastics; but, protection is provided selected testrubbers.

PART IA: FORMULA—PERACID COMPONENT HIGH POAA—LOW H202 PEPACID FORMULAKX-6091

GM/ ITEM RAW MATERIAL WT % 10000 10 Acetic Acid 78.00 7800.00 20Hydrogen Peroxide 35% 21.00 2100.00 30 Dequest ™ 2010 (60%) 1.00 100.00Total 100.00 10000.00

Mixing Instructions:

Batch was prepared by direct weighing on Mettler PM 16 Top LoadingBalance into a 5 gal HMW/HDPE (high molecular weight/high densitypolypropylene) pail. The batch was mixed for 65 minutes using a labmixer equipped with a plastic coated stir rod and blade.

PART IB: FORMULA - ADMIXTURE OF IA AND BUFFER COMPONENT FORMULAS A, B,C, D CORROSION STUDY USE DILUTIONS (A) (B) (C) (D) GM/ GM/ GM/ GM/ ITEMMaterial WT % 4500 WT % 4500 WT % 4500 WT % 4500 10 Deionized 99.105564459.75 90.66311 4079.84 99.55756 4480.09 95.57511 4300.88 Water 20Trisodium 0.45200 20.41 4.91200 221.04 Phosphate Anhyd. Gran. 30 KX-60910.44244 19.91 4.42489 199.12 0.44244 19.91 4.42489 199.12 (11.3% POAA)Total 100.00000 4500.07 100.00000 4500.00 100.00000 4500.00 100.000004500.00 THEORETICAL VALUES ppm pH ppm pH ppm pH ppm pH POAA 500 6.005000 6.00 500 3.00 5000 2.50 INSTRUCTIONS Add Trisodium PhosphateAnhydrous Granules (item 20) by wt. to weighed amount of DI water andstir with Lab mixer until dissolved. Add (item 30) by wt. to bufferedwater and final mix 2 min. RESULTS: (A) - pH = 6.02 (B) - pH = 5.99(C) - pH = 2.96 (D) - pH = 2.35

PART II: CORROSION - METALS 14 day Compatibility Test of 15 differentmaterials tested against four different Test Solutions at 50° C. withthe test solutions are changed daily. Material Initial Wt. Final Wt.Test item Test Solution METALS (gms) (gms) TWL CWL AWL mpy  1 (A) 500ppm POAA/Buffered 316 SS 23.5792 23.5791 0.0001 0.0001 0.0000 0.0000  5(B) 5000 ppm POAA/Buffered 316 SS 23.5194 23.5193 0.0001 0.0001 0.00000.0000  9 (C) 500 ppm POAA only 316 SS 23.5764 23.5762 0.0002 0.00010.0000 0.0031 13 (D) 5000 ppm POAA only 316 SS 23.5690 23.5689 0.00010.0001 0.0000 0.0000 17 CONTROL 316 SS 23.5846 23.5845 0.0001 0.0001  2(A) 500 ppm POAA/Buffered 304 SS 17.9651 17.9650 0.0001 0.0000 0.00010.0031  6 (B) 5000 ppm POAA/Buffered 304 SS 17.9326 17.9323 0.00030.0000 0.0030 0.0938 10 (C) 500 ppm POAA only 304 SS 17.9795 17.97930.0002 0.0000 0.0002 0.0063 14 (D) 5000 ppm POAA only 304 SS 17.999317.9992 0.0001 0.0000 0.0001 0.0031 18 CONTROL 304 SS 18.1102 18.11020.0000 0.0000  3 (A) 500 ppm POAA/Buffered 7075 12.8716 12.8685 0.00310.0002 0.0029 0.2412 Aluminum  7 (B) 5000 ppm POAA/Buffered 7075 12.757512.7336 0.0239 0.0002 0.0237 1.9712 Aluminum 11 (C) 500 ppm POAA only7075 12.8651 12.8392 0.0259 0.0002 0.0257 2.1376 Aluminum 15 (D) 5000ppm POAA only 7075 12.8718 12.7439 0.1279 0.0002 0.1277 10.6213 Aluminum19 CONTROL 7075 12.4899 12.4897 0.0002 0.0002 Aluminum  4 (A) 500 ppmPOAA/Buffered 260 Brass 26.4108 26.3763 0.0345 0.0004 0.0341 0.9779  8(B) 5000 ppm POAA/Buffered 260 Brass 26.4211 26.3307 0.0904 0.00040.0900 2.5809 12 (C) 500 ppm POAA only 260 Brass 26.6471 25.6695 0.97760.0004 0.9772 28.0233 16 (D) 5000 ppm POAA only 260 Brass 26.494918.9759 7.5190 0.0004 7.5186 215.6118 20 CONTROL 260 Brass 26.435226.4348 0.0004 0.0004 Test Material item Test Solution METALS VisualObservations  1 (A) 500 ppm POAA/Buffered 316 SS Smooth, shiny silvercolored material like control  5 (B) 5000 ppm POAA/Buffered 316 SSSmooth, shiny silver colored material like control  9 (C) 500 ppm POAAonly 31 6 SS Smooth, shiny silver colored material like control 13 (D)5000 ppm POAA only 316 SS Smooth, shiny silver colored material likecontrol 17 CONTROL 316 SS Smooth, shiny silver colored material  2 (A)500 ppm POAA/Buffered 304 SS Smooth, shiny silver colored material likecontrol  6 (B) 5000 ppm POAA/Buffered 304 SS Smooth, shiny silvercolored material like control 10 (C) 500 ppm POAA only 304 SS Smooth,shiny silver colored material like control 14 (D)5000 ppm POAA only 304SS Smooth, shiny silver colored material like control 18 CONTROL 304 SSSmooth, shiny silver colored material  3 (A) 500 ppm POAA/Buffered 7075Aluminum A slt. duller, slt. whiter than control, silver material  7 (B)5000 ppm POAA/Buffered 7075 Aluminum A very dull, smokey brown coloredmaterial 11 (C) 500 ppm POAA only 7075 Aluminum A dull, whitish graycolored material 15 (D) 5000 ppm POAA only 7075 Aluminum A very dull,very whitish gray colored material 19 CONTROL 7075 Aluminum A slt. dull,silver colored material  4 (A) 500 ppm POAA/Buffered 260 Brass A mixtureof dull gold & pink area colored material  8 (B) 5000 ppm POAA/Buffered260 Brass A dull, gold colored material with patches of pink 12 (C) 500ppm POAA only 260 Brass A darker dull gold colored material with pinkareas 16 (D) 5000 ppm POAA only 260 Brass A sparkling grainy goldcolored material 20 CONTROL 260 Brass A smooth, shiny, gold coloredmaterial

KX-6091 CORROSION STUDY CALCULATION DATA DENSITY AREA in inches squared4 Metals 316 Stainless Steel 7.98 6.5 304 Stainless Steel; 7.94 6.4 7075Aluminum 2.81 6.8 260 Brass 8.5 6.52 Time & Temp Tested 14 days at 50°C. mpy = (534,000 * AWL)/(A * T * D) (A) = Area (see above) (T) = Time(336 hrs) (D) = Density (see above) AWL = TWL − CWL TWL = Pre-testingweight − Post-testing weight CWL = Pre-testing weight of control −Post-testing weight of control mpy = mils per year

PART III: CORROSION - PLASTICS Analytical - Observations KX-6091CORROSION STUDY 14 day Compatibility Test of 15 different materialstested against four different Test Solutions at 50° C. with the testsolutions are changed daily. Initial Initial Test Material Initial Wt.Initial Ht. Width Thick Final Wt. % Weight item Test Solution PLASTICS(gms) (inches) (Inches) (inches) (gms) Change 21 (A) 500 ppmPolyurethane 3.8348 2.996 0.506 0.128 3.8360 0.0313 POAA/Buffered 27 (B)5000 ppm Polyurethane 3.8379 2.996 0.502 0.129 3.8385 0.0156POAA/Buffered 33 (C) 5000 ppm POAA Polyurethane 3.8385 2.999 0.505 0.1283.8418 0.0860 only 39 (D) 5000 ppm Polyurethane 3.8151 2.995 0.504 0.1273.7411 −1.9397 POAA only 45 CONTROL Polyurethane 3.8286 2.996 0.5050.128 3.8200 −0.2248 22 (A) 500 ppm Polyethylene 1.3741 2.991 0.5050.066 1.3736 −0.0364 POAA/Buffered 28 (B) 5000 ppm Polyethylene 1.36762.991 0.505 0.064 1.3675 −0.0073 POAA/Buffered 34 (C) 500 ppm POAAPolyethylene 1.3541 2.992 0.504 0.065 1.3541 0.0000 only 40 (D) 5000 ppmPolyethylene 1.3586 2.995 0.504 0.066 1.3593 0.0515 POAA only 46 CONTROLPolyethylene 1.3668 2.991 0.504 0.068 1.3667 −0.0073 23 (A) 500 ppmPolypropylene 1.3792 3.002 0.504 0.066 1.3792 0.0000 POAA/Buffered 29(B) 5000 ppm Polypropylene 1.3774 2.998 0.503 0.065 1.3775 0.0073POAA/Buffered 35 (C) 500 ppm POAA Polypropylene 1.3793 2.998 0.504 0.0651.3796 0.0218 only 47 CONTROL Polypropylene 1.3812 2.997 0.503 0.0651.3811 −0.0072 24 (A) 500 ppm Polyvinyl 2.1801 3.002 0.505 0.066 2.18430.1927 POAA/Buffered Chloride 30 (B) 5000 ppm Polyvinyl 2.2005 2.9970.505 0.066 2.2041 0.1636 POAA/Buffered Chloride 36 (C) 500 ppm POAAPolyvinyl 2.1734 2.998 0.505 0.065 2.1777 0.1978 only Chloride 42 (D)5000 ppm Polyvinyl 2.1590 2.998 0.505 0.065 2.1625 0.1621 POAA onlyChloride 48 CONTROL Polyvinyl 2.2048 2.999 0.505 0.056 2.2037 −0.0499Chloride 25 (A) 500 ppm ABS 1.4724 2.995 0.507 0.061 1.4762 0.2581POAA/Buffered 31 (B) 5000 ppm ABS 1.5167 3.003 0.507 0.063 1.5201 0.2242POAA/Buffered 37 (C) 500 ppm POAA ABS 1.5082 3.000 0.507 0.062 1.51320.3315 only 43 (D) 5000 ppm ABS 1.4971 2.995 0.505 0.062 1.5047 0.5076POAA only 49 CONTROL ABS 1.4822 2.995 0.507 0.062 1.4813 −0.0607 26 (A)500 ppm Polyacetal 4.4596 3.003 0.507 0.133 4.5033 0.9799 POAA/Buffered32 (B) 5000 ppm Polyacetal 4.3970 3.003 0.507 0.131 4.4302 0.7551POAA/Buffered 38 (C) 500 ppm POAA Polyacetal 4.4967 3.004 0.506 0.1344.5441 1.0092 only 44 (D) 5000 ppm Polyacetal 4.3832 3.003 0.507 0.1314.4264 0.9856 POAA only 50 CONTROL Polyacetal 4.4498 3.002 0.506 0.1334.4454 −0.0989 Final % Final Test Final Ht. % Height Width Width Thick %Thick item (inches) Change (inches) Change (inches) Changes 21 2.9960.0000 0.507 0.1976 0.128 0.0000 27 2.998 0.0668 0.502 0.0000 0.128−0.7752 33 3.004 0.1567 0.505 −0.1976 0.127 −0.7813 39 3.061 2.20370.509 0.9921 0.125 −1.5748 45 2.993 −0.1001 0.504 −0.1980 0.128 0.000022 2.991 0.0000 0.504 −0.1980 0.066 0.0000 28 2.991 0.0000 0.505 0.00000.065 1.5625 34 2.991 −0.0334 0.502 −0.3968 0.065 0.0000 40 2.994−0.0334 0.502 −0.3968 0.066 0.0000 46 2.989 −0.0669 0.504 0.0000 0.0680.0000 23 3.001 −0.0333 0.503 −0.1984 0.067 1.5152 29 2.999 0.0334 0.5030.0000 0.066 1.5385 35 2.998 0.0000 0.503 −0.1984 0.065 0.0000 47 2.9970.0000 0.503 0.0000 0.065 0.0000 24 3.002 0.0000 0.506 0.1980 0.065−1.5152 30 2.997 0.0000 0.506 0.1980 0.066 0.0000 36 2.998 0.0000 0.5050.0000 0.065 0.0000 42 2.997 −0.0334 0.505 0.0000 0.065 0.0000 48 2.998−0.0333 0.505 0.0000 0.056 0.0000 25 2.999 0.1336 0.508 0.1972 0.0610.0000 31 3.006 0.0999 0.506 −0.1972 0.063 0.0000 37 3.004 0.1333 0.5080.1972 0.062 0.0000 43 3.000 0.1669 0.510 0.9901 0.062 0.0000 49 2.9950.0000 0.508 0.1972 0.062 0.0000 26 3.010 0.2331 0.508 0.1972 0.1340.7519 32 3.009 0.1998 0.507 0.0000 0.132 0.7634 38 3.014 0.3329 0.5080.3953 0.135 0.7463 44 3.012 0.2997 0.508 0.1972 0.132 0.7634 50 3.000−0.0666 0.506 0.0000 0.133 0.0000 Test Material item Test SolutionPLASTICS Visual Observations 21 (A) 500 ppm POAA/Buffered PolyurethaneDull opaque orange material with semi-transparent boarder 27 (B) 5000ppm POAA/Buffered Polyurethane Dull opaque orange material withsemi-transparent boarder and slt. tacky 33 (C) 500 ppm POAA onlyPolyurethane Dull darker opaque orange material with semi-transparentboarder and slt. tacky 39 (D) 5000 ppm POAA only Polyurethane Very darkorange, very tacky, completely opaque material that stuck to dryingsurface resulting in loss of material 45 CONTROL Polyurethane A dull,dirty, slt. yellow tinted, semi-transparent material 22 (A) 500 ppmPOAA/Buffered Polyethylene Slt. whiter material than control 28 (B) 5000ppm POAA/Buffered Polyethylene Slt. whiter material than control 34 (C)500 ppm POAA only Polyethylene Slt. whiter material than control 40 (D)5000 ppm POAA only Polyethylene Slt. whiter material than control 46CONTROL Polyethylene A dull, grayish white material 23 (A) 500 ppmPOAA/Buffered Polypropylene A white filmy, faintly transparent, morecloudy material than control 29 (B) 5000 ppm POAA/Buffered PolypropyleneA white filmy, faintly transparent, more cloudy material than control 35(C) 500 ppm POAA only Polypropylene A white heavy filmed, faintlytransparent, more cloudy material than control 41 (D) 5000 ppm POAA onlyPolypropylene A white filmy, faintly transparent, more cloudy materialthan control 47 CONTROL Polypropylene A dull gray, semi-transparentmaterial 24 (A) 500 ppm POAA/Buffered Polyvinyl Slt. less shiny and slt.less dark gray material than control Chloride 36 (C) 500 ppm POAA onlyPolyvinyl A dull med. gray material Chloride 42 (D) 5000 ppm POAA onlyPolyvinyl A dull light to medium gray material Chloride 48 CONTROLPolyvinyl A dark, shiny gray material Chloride 25 (A) 500 ppmPOAA/Buffered ABS A slt. dull, whiter material than control 31 (B) 5000ppm POAA/Buffered ABS A slt. dull, whiter material than control 37 (C)500 ppm POAA only ABS A slt. dull, much whiter white material thancontrol 43 (D) 5000 ppm POAA only ABS A slt. dull bright white material49 CONTROL ABS A slt. dull, vanilla white material 26 (A) 500 ppmPOAA/Buffered Polyacetal A dull, cleaner white appearance than control32 (B) 5000 ppm POAA/Buffered Polyacetal A dull, cleaner whiteappearance than control 38 (C) 500 ppm POAA only Polyacetal A dull,cleaner white appearance than control 44 (D) 5000 ppm POAA onlyPolyacetal A dull, cleaner white appearance than control 50 CONTROLPolyacetal A dull, dirty white material

PART IV: CORROSION - RUBBERS Analytical - Observations KX-6091 CORROSIONSTUDY 14 day Compatibility Test of 15 different materials tested againstfour different Test Solutions at 50° C. with the test solutions arechanged daily. Test Material Initial Wt. Initial Ht. Initial WidthInitial thick Final Wt. % Weight item Test Solution RUBBERS (gms)(inches) (inches) (inches) (gms) Change 51 (A) 500 ppm Silicone 14.27242.930 0.928 0.254 14.2553 −0.1198 POAA/Buffered 56 (B) 5000 ppm Silicone15.5707 2.999 1.007 0.249 15.5665 −0.0270 POAA/Buffered 61 (C) 500 ppmSilicone 15.6958 3.013 0.995 0.252 15.7755 0.5078 POAA only 66 (D) 5000ppm Silicone 15.1443 2.977 0.994 0.246 15.3760 1.5299 POAA only 71CONTROL Silicone 15.6702 2.970 1.001 0.253 15.6417 −0.1819 52 (A) 500ppm Butyl 1.9074 2.999 0.507 0.069 1.9852 4.0789 POAA/Buffered 57 (B)5000 ppm Butyl 1.9082 2.999 0.505 0.069 1.9263 0.9485 POAA/Buffered 62(C) 500 ppm Butyl 1.9026 2.996 0.505 0.068 2.0729 8.9509 POAA only 67(D) 5000 ppm Butyl 1.9097 2.998 0.507 0.069 2.2216 16.3324 POAA only 72CONTROL Butyl 1.9001 2.998 0.507 0.069 1.8939 −0.3263 53 (A) 500 ppmVison 23.3725 3.057 1.031 0.248 23.4407 0.2918 POAA/Buffered 58 (B) 5000ppm Vison 21.3847 2.984 1.014 0.237 21.4843 0.5598 POAA/Buffered 68 (D)5000 ppm Vison 22.4157 2.964 1.012 0.251 23.7728 6.0542 POAA only 73CONTROL Vison 22.0694 2.988 1.012 0.244 22.0584 −0.0498 54 (A) 500 ppmEPDM 17.0399 3.042 1.005 0.277 17.1763 0.8005 POAA/Buffered 59 (B) 5000ppm EPDM 16.9577 3.033 1.006 0.278 17.2265 1.5851 POAA/Buffered 64 (C)500 ppm EPDM 16.9824 3.059 1.015 0.275 16.9653 −0.1007 POAA only 69 (D)5000 ppm EPDM 17.4875 2.985 1.072 0.274 17.9757 2.7917 POAA only 74CONTROL EPDM 16.7254 2.964 1.016 0.278 16.6918 −0.2009 55 (A) 500 ppmBUNA N 15.8678 2.960 1.006 0.242 16.3169 2.8303 POAA/Buffered 80 (B)5000 ppm BUNA N 15.9576 2.980 1.020 0.240 16.4275 2.9447 POAA/Buffered85 (C) 500 ppm BUNA N 16.2737 2.977 1.016 0.246 18.9478 4.1423 POAA only70 (D) 5000 ppm BUNA N 15.8516 2.956 1.014 0.242 16.5043 4.1176 POAAonly 75 CONTROL BUNA N 16.0735 2.936 1.107 0.247 16.0328 −0.2532 TestFinal Ht. % Height Final Width % Width Final Thick % Thick item (inches)Change (inches) Change (inches) Change 51 2.930 0.0000 0.933 0.53880.254 0.0000 56 2.995 −0.1334 1.008 0.0993 0.249 0.0000 61 3.019 0.19911.004 0.9045 0.252 0.0000 66 3.003 0.6734 1.005 1.1066 0.249 1.2195 712.970 0.0000 1.013 1.1988 0.254 0.3953 52 3.008 0.3001 0.507 0.00000.071 2.8986 57 3.008 0.3001 0.505 0.0000 0.069 0.0000 62 3.017 0.70090.513 1.5842 0.075 10.2941 67 3.029 1.0340 0.494 −2.5841 0.078 13.043572 2.998 −0.0867 0.504 −0.5917 0.069 0.0000 53 3.071 0.4580 1.033 0.19400.248 0.0000 58 2.998 0.4692 1.025 1.0848 0.238 0.4219 68 3.064 3.37381.053 4.0514 0.260 3.5857 73 2.991 0.1004 1.012 0.0000 0.244 0.0000 543.053 0.3616 1.009 0.3980 0.285 2.8881 59 3.036 0.0989 1.012 0.59640.285 2.5180 64 3.068 0.2942 1.012 −0.2956 0.282 2.5455 69 3.020 1.17251.079 0.6530 0.284 3.6496 74 2.959 −0.1687 1.015 −0.0984 0.278 0.0000 552.970 0.3378 1.012 0.5964 0.247 2.0661 80 2.989 0.3020 1.019 −0.09800.246 2.5000 85 2.992 0.5039 1.024 0.7874 0.259 5.2846 70 2.956 0.00001.029 1.4793 0.264 9.0909 75 2.937 0.0341 1.014 −0.2950 0.247 0.0000Test Material item Test Solution RUBBERS Visual Observations 51 (A) 500ppm POAA/Buffered Silicone A dull, med. - dark orange material similarto control 56 (B) 5000 ppm POAA/Buffered Silicone A dull, med. - darkorange material similar to control 61 (C) 500 ppm POAA only Silicone Adull, med. - dark orange material similar to control 66 (D) 5000 ppmPOAA only Silicone A dull, med. - dark orange material similar tocontrol 71 CONTROL Silicone A dull, med. - dark orange material 52 (A)500 ppm POAA/Buffered Butyl A dull black material with slt. tacky, slt.rough surface that stuck to drying surface resulting in loss of material57 (B) 5000 ppm POAA/Buffered Butyl A dull black material with very slt.tacky, smooth surface 62 (C) 500 ppm POAA only Butyl A black materialwith tacky, dull, rough surface that stuck to drying surface resultingin loss of material 67 (D) 5000 ppm POAA only Butyl A dull blackmaterial with very tacky, very rough, surface that stuck to dryingsurface resulting in loss of material 53 (A) 500 ppm POAA/Buffered VisonA dull, charcoal black material with smooth surface 58 (B) 5000 ppmPOAA/Buffered Vison A dull, charcoal black material with smooth surface63 (C) 500 ppm POAA only Vison A dull, charcoal black material with slt.rough surface 68 (D) 5000 ppm POAA only Vison A dull, charcoal blackmaterial with slt. rough surface 73 CONTROL Vison A dull, charcoal blackmaterial with smooth surface 54 (A) 500 ppm POAA/Buffered EPDM A dull,black material with slt. rough surface 59 (B) 5000 ppm POAA/BufferedEPDM A dull, black material with slt. blistered surface 64 (C) 500 ppmPOAA only EPDM A dull, black material with slt. rough surface 69 (D)5000 ppm POAA only EPDM A dull black material with slt. rough surfacecontaining a large blister 74 CONTROL EPDM A dull, black material withsmooth surface 55 (A) 500 ppm POAA/Buffered BUNA N A dull, (darker thancontrol) black material with slt. rough surface 60 (B) 5000 ppmPOAA/Buffered BUNA N A dark black material with very slt. shiny, fairlysmooth surface 65 (C) 500 ppm POAA only BUNA N A dark black materialwith very slt. shiny, slt. blistered surface 70 (D) 5000 ppm POAA onlyBUNA N A dark black material with very slt. shiny, blistered surface 75CONTROL BUNA N A dull, grayish black material with smooth surface

I. Tuberculocidal Efficacy US Method

The peracetic acid product was tested against Mycobacterium bovis (BCG)using the AOAC Confirmatory Test with product concentrations as listedbelow. The product was diluted in buffer to achieve the pH 6 prior totest. The diluent tested was either tap or distilled water. Testexposure time was 10 minutes. A result of ten no growth tubes per tentubes tested is required for a passing result. Conclusion: successfultuberculocidal results were achieved a product concentrations as low as1000 ppm POAA.

Number of no growth tubes/ Product Concentration^(a) number of tubestested^(b) 1000 ppm POAA 10/10 - pass 2000 ppm POAA 10/10 - pass 3000ppm POAA 10/10 - pass 4000 ppm POAA 10/10 - pass 5000 ppm POAA 10/10 -pass ^(a)Diluent was tap or distilled water with pH adjusted to 6.^(b)Test results reflect data achieved in three test media,Proskauer-Beck, Kirshners and Middlebrook.

II. Suspension Test—Olympus Method

We have completed the suspension test as requested with the Olympusprocedure versus Bacillus subtilis. The product was diluted in buffer toachieve the pH 6 prior to test. The diluent tested was tap water. Testexposure times are listed below. The data are represented as logreduction of bacterial numbers. Note: the spores were counted after theheat shock treatment, although the test was conducted on a non-heattreated bacterial suspension. Conclusion significant log reductions inmicrobial numbers were achieved within 10 minutes using 500 ppm POAA.Additional product concentration or exposure time did not increase theefficacy of the product.

Bacillus subtilis Log Reduction at 20° C. (ppm POAA) 1500 ppm 2000 ppmExposure time (Henkel-Ecolab (Ecolab test (minutes) 250 ppm 500 ppm 1000ppm test only) only)  5 minutes 4.55 6.13 9.48 7.70 9.78 10 minutes 7.989.78 9.78 7.68 9.78 20 minutes 9.48 9.78 9.78 7.71 9.78 60 minutes 9.489.78 9.78 7.74 9.78 Neutralization control 0.10^(A) Total inoculum 3.4 ×10⁸ cfu/ml 6.0 × 10⁹ cfu/ml Spore inoculum 9.0 × 10⁶ cfu/ml 3.3 × 10⁵cfu/ml ^(A)Neutralizer is 1 % sodium thiosulfate and is effective inthis test procedure for chemical neutralization of the test substance.

III. Carrier Test—Olympus Method

We have completed the carrier test as requested using the Olympusprocedure versus Bacillus subtilis and Mycobacterium terrae. The productwas diluted in buffer to achieve the pH 6 prior to test The diluenttested was tap water. Test exposure times are listed below. Note: thespores were counted after the heat shock treatment although the test wasconducted on a non-heat treated bacterial suspensions. Conclusion:successful results achieved using 250 ppm POAA within five minutesexposure against both subtilis and Mycobacterium terrae. Additionalproduct concentration or exposure time did not increase the efficacy ofthe product.

250 ppm 1000 ppm 2500 ppm 5000 ppm Exposure time CARRIER^(A) CARRIERCARRIER CARRIER (minutes) RESULTS A^(B) B^(C) RESULTS A B RESULTS A BRESULTS A B Bacillus subtilis at 20° C. (ppm POAA)  0 minutes 0/2 2.3 ×10⁴ 1.9 × 10³  5 minutes 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 10minutes 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 20 minutes 2/2 <1 <1 2/2<1 <1 2/2 <1 <1 2/2 <1 <1 60 minutes 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2<1 <1 Mycobacterium terrae at 20° C. (ppm POAA)  0 minutes 0/2 3.2 × 10³2.1 × 10⁴  5 minutes 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 10 minutes2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 20 minutes 2/2 <1 <1 2/2 <1 <12/2 <1 <1 2/2 <1 <1 60 minutes 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1 2/2 <1 <1^(A)Number of negative carriers per number of carriers tested. ^(B)PlateA is the average cfu/ml of product plus neutralizer mixture. ^(C)Plate Bis the average cfu/ml of stripper. ^(D)Neutralizer is 1% sodiumthiosulfate and is effective in this test procedure for chemicalneutralization of the test substance.

IV. Sporicidal Efficacy—US Method

The peracetic acid product was tested against Clostridium sporogenesusing the AOAC Sporicdal Activity of Disinfectants Test with productconcentrations as listed below. The product was diluted in buffer toachieve the pH 6 prior to test The diluent tested was tap water. Testexposure time was 3, 4 or 6 hours. A result of twenty no growth tubesper twenty tubes tested is required for a passing result. Conclusion:successful results were achieved at 5000 ppm POM with an exposure timeof 6 hours.

Number of no growth tubes/ number of tubes tested^(b) Product ExposureSecondary Concentration^(a) Time Primary Subculture Subculture 4000 ppmPOAA 3 hours 20/20  0/20 4 hours 20/20  1/20 6 hours 19/20 20/20 5000ppm POAA 3 hours 19/20  6/20 4 hours 20/20 17/20 6 hours 20/20 20/207000 ppm POAA 3 hours 20/20 10/20 4 hours 20/20 11/20 6 hours 20/2020/20 ^(a)Diluent was tap or distilled water with pH adjusted to 6.^(b)Test results reflect data achieved in three test media,Proskauer-Beck, Kirshners and Middlebrook after heat-shock treatment andreincubation for 72 hours.

OBJECTIVE:

The objective of this analysis was to evaluate the effect of hydrogenperoxide and acetic acid concentration on the sporicidal efficacy of 150ppm peracetic acid at 40° C.

TEST METHOD:

Ecolab Microbiological Services SOP CB021-04; Rate of Kill AntimicrobialEfficacy. Following exposure to the formula and subsequentneutralization, spores were heat shocked for 13 minutes at 80° C. beforeplating.

METHOD PARAMETERS:

Test Substances: Each formula was prepared using a “stock” POAA material(34.1% POAA, 7.13% H₂O₂ and 36.1% acetic acid—Aldrich Chemical) toachieve 150 ppm POAA. H₂O₂ or acetic acid was then added as needed.Please refer to the data sheet attached to this report for preparationinformation. Since chemical analyses of solutions prepared exactly likethose prepared for this study were done previously, and concentrationswere found to be accurate, additional chemical analysis for this studywas not performed (see MSR #960351, J. Hilgren).

Chemical Properties of Each Test Formula Theoretical TheoreticalTheoretical Formula ppm POAA ppm H₂O₂ ppm Acetic Acid pH A 150 31 1593.75 B 150 31 309 3.67 C 150 275 159 3.75 D 150 275 309 3.68 E 150 529159 3.77 F 150 329 309 3.68

Test System: Bacillus cereus spore crop N1009

Test Temperature: 40° C.

Exposure Times: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 hours

Neutralizer: Fluid Thioglycollate Medium

Plating Media: Dextrose Tryptone Agar

Incubation: 32° C. for 48 hours

RESULTS:

Inoculum Numbers Inoculum Test Replicate (CFU/mL) Average Organism 1 2 3(CFU/mL) B. cereus Spores 30 × 10⁶ 26 × 10⁶ 26 × 10⁶ 2.7 × 10⁷ Reductionof B. cereus Spores at 40° C. Exposure Time Formula (hours) Survivors(CFU/mL) Log Reduction A 0.5 <1.0 × 10¹ >6.43 Low Acetic, 1.0 <1.0 ×10¹ >6.43 Low H₂O₂ 1.5 <1.0 × 10¹ >6.43 2.0 <1.0 × 10¹ >6.43 2.5 <1.0 ×10¹ >6.43 3.0 <1.0 × 10¹ >6.43 3.5 <1.0 × 10¹ >6.43 B 0.5 <1.0 ×10¹ >6.43 High Acetic, 1.0 <1.0 × 10¹ >6.43 Low H₂O₂ 1.5 <1.0 ×10¹ >6.43 2.0 <1.0 × 10¹ >6.43 2.5 <1.0 × 10¹ >6.43 3.0 <1.0 × 10¹ >6.433.5 <1.0 × 10¹ >6.43 C 0.5 1.7 × 10⁷ 0.20 Low Acetic, 1.0 6.0 × 10⁶ 0.65Medium H₂O₂ 1.5 2.5 × 10⁶ 1.03 2.0 9.0 × 10⁵ 1.48 2.5 2.1 × 10⁵ 2.11 3.06.0 × 10⁴ 2.65 3.5 1.5 × 10⁴ 3.26 D 0.5 1.5 × 10⁷ 0.26 High Acetic, 1.04.9 × 10⁶ 0.74 Medium H₂O₂ 1.5 2.2 × 10⁶ 1.09 2.0 4.6 × 10⁵ 1.77 2.5 1.2× 10⁵ 2.35 3.0 3.1 × 10⁴ 2.94 3.5 1.1 × 10⁴ 3.39 E 0.5 1.5 × 10⁷ 0.26Low Acetic, 1.0 5.1 × 10⁶ 0.72 High H₂O₂ 1.5 1.4 × 10⁶ 1.29 2.0 3.1 ×10⁵ 1.94 2.5 3.4 × 10⁴ 2.90 3.0 4.0 × 10³ 3.83 3.5 5.6 × 10² 4.68 F 0.51.4 × 10⁷ 0.29 High Acetic, 1.0 4.7 × 10⁶ 0.76 High H₂O₂ 1.5 1.7 × 10⁶1.20 2.0 4.3 × 10⁵ 1.80 2.5 3.3 × 10⁴ 2.91 3.0 5.0 × 10³ 3.73 3.5 8.1 ×10² 4.52

A graphical representation of the reduction of B. cereus spores at 40°C. is presented in FIG. 3. The lower limit of detection for the testprocedure was 10 C.F.U./mL.

CONCLUSIONS:

The sporicidal activity of 150 ppm POAA at 40° C. against Bacilluscereus spores was most effective when in the presence of relatively lowconcentrations of H₂O₂ (≈330 ppm as in Formulas A and B). Reduced B.cereus sporicidal efficacy was observed using POAA with the medium andhigh concentrations of H₂O₂ (≈160 and 300 ppm as in Formulas C throughF).

OBJECTIVE:

The objective of this analysis was to evaluate the effect of hydrogenperoxide and acetic acid concentration on the sporicidal efficacy of 150ppm peracetic acid at 60° C.

TEST METHOD:

Ecolab Microbiological Services SOP CB021-04; Rate of Kill AntimicrobialEfficacy. Following exposure to the formula and subsequentneutralization, spores were heat shocked for 13 minutes at 80° C. beforeplating.

METHOD PARAMETERS:

Test Substances: Each formula was prepared using a “stock” POAA material(34.1% POAA, 7.13% H₂O₂ and 36.1% acetic acid—Aldrich Chemical) toachieve 150 ppm POAA. H₂O₂or acetic acid was then added as needed.Please refer to the data sheet attached to this report for theoreticalconcentrations and preparation information.

Formula Properties (≈ 2 Hours Post Preparation/ After 40 min. at 60° C.)Formula ppm POAA ppm H₂O₂ ppm Acetic Acid pH A 147/144 31/33 174/1663.76/3.67 B 145/144 33/37 346/346 3.71/3.55 C 151/148 277/281 141/1433.79/3.69 D 151/151 283/280 301/291 3.70/3.60 E 157/154 526/514 136/1483.81/3.71 F 160/159  533/240* 293/324 3.71/3.62 *No obvious error inanalysis was detected, but the result remains in question.

Test System: Bacillus cereus spore crop N1009

Test Temperature: 60° C.

Exposure Times: 10, 15, 20, 25, 30 and 40 minutes

Neutralizer: Fluid Thioglycollate Medium

Plating Media: Dextrose Tryptone agar

Incubation: 32° C. for 48 hours

Inoculum Numbers Inoculum Test Replicate (CFU/mL) Average Organism 1 2 3(CFU/mL) B. cereus Spores 28 × 10⁶ 22 × 10⁶ 29 × 10⁶ 2.6 × 10⁷ Reductionof B. cereus Spores at 60° C. Exposure Time Formula (hours) Survivors(CFU/mL) Log Reduction A 10 <1.0 × 10¹ >6.41 Low Acetic, 15 <1.0 ×10¹ >6.41 Low H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41 B 10 <1.0 × 10¹ >6.41 High Acetic, 15 <1.0× 10¹ >6.41 Low H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41 C 10 4.1 × 10⁴ 2.80 Low Acetic, 15 2.0 ×10² 5.11 Medium H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41 D 10 2.6 × 10⁴ 3.00 High Acetic, 15 7.0 ×10¹ 5.57 Medium H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41 E 10 2.4 × 10⁴ 3.03 Low Acetic, 15 2.4 ×10² 5.03 High H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41 F 10 1.1 × 10⁴ 3.37 High Acetic, 15 7.0 ×10¹ 5.57 High H₂O₂ 20 <1.0 × 10¹ >6.41 25 <1.0 × 10¹ >6.41 30 <1.0 ×10¹ >6.41 40 <1.0 × 10¹ >6.41

A graphical representation of the reduction of B. cereus spores at 60°C. It is shown in FIG. 2. The lower limit of detection for the testprocedure was 10 C.F.U./mL.

CONCLUSIONS

The sporicidal activity of 150 ppm POAA at 60° C. against Bacilluscereus spores was most effective when in the presence of relatively lowconcentrations of H₂O₂ (≈160 and 300 ppm as in Formulas C through F).

Further testing using Formulas A-F will be conducted at 20° C. todetermine the effect of H₂O₂ and acetic acid concentration on sporicidalefficacy of POAA at low temperature.

OBJECTIVE:

The objective of this analysis was to evaluate the effect of hydrogenperoxide, octanoic acid and peroctanoic acid concentration on thesporicidal efficacy of 150 ppm peracetic acid at 40° C.

TEST METHOD:

Ecolab Microbiological Services SOP CB021-04; Rate of Kill AntimicrobialEfficacy. Following exposure to the formula and subsequentneutralization, spores were heat shocked for 13 minutes at 80° C. beforeplating.

METHOD PARAMETERS:

Test Substances: Each formula was prepared using a “stock” POAA material(33.5% POAA, 7.03% H₂O₂ and 37.2% acetic acid—Aldrich Chemical) and a“stock” octanoic/peroctanoic material (11.4% octanoic, 3.4% POOA 10.29%POAA, 3.70% H₂O₂—Falcon 15). Hydrogen peroxide, octanoic acid orperoctanoic acid were then added as needed. Please refer to the datasheet attached to this report for preparation information. Prior to thisstudy, chemical analyses of formulas exactly like those used for thisstudy were conducted to determine if ingredient concentrations wereclose to theoretical and if they were stable over the duration of theefficacy test. Results showed ingredient concentrations to correlatewith theoretical and to be stable.

Chemical Properties of Each Test Formula Theoretical TheoreticalTheoretical Theoretical Theoretical Formula ppm POAA ppm H₂O₂ ppm AA ppmPOOA ppm OA pH 1 149 36 282 12 39 3.65 2 149 529 282 12 39 3.62 3 149 36282 50 39 3.64 4 149 529 282 50 39 3.63 5 149 36 282 12 138 3.64 6 149529 282 12 138 3.63 7 149 36 282 50 138 3.64 8 149 529 282 50 138 3.65

Test System: Bacillus cereus spore crop N1009

Test Temperature: 40° C.

Exposure Times: 5, 10, 15, 20, 25 and 30 minutes

Neutralizer: Fluid Thioglycollate Medium

Plating Medium: Dextrose Tryptone Agar

Incubation; 32° C. for 48 hours

RESULTS

Inoculum Numbers Inoculum Test Replicate (CFU/mL) Average Organism 1 2 3(CFU/mL) B. cereus Spores 56 × 10⁶ 42 × 10⁶ 35 × 10⁶ 4.4 × 10⁷ Reductionof B. cereus Spores at 40° C. Exposure Time Formula (hours) Survivors(CFU/mL) Log Reduction 1 5 3.0 × 10¹ 6.17 Low H₂O₂, 10 <1.0 × 10¹ >6.64Low POOA, 15 <1.0 × 10¹ >6.64 Low OA 20 <1.0 × 10¹ >6.64 25 <1.0 ×10¹ >6.64 30 <1.0 × 10¹ >6.64 2 5 6.4 × 10⁶ 0.84 High H₂O₂, 10 4.3 × 10⁶1.01 Low POOA, 15 1.8 × 10⁶ 1.39 Low OA 20 4.0 × 10⁵ 2.04 25 1.2 × 10⁵2.56 30 8.1 × 10⁴ 2.73 3 5 <1.0 × 10¹ >6.64 Low H₂O₂, 10 <1.0 ×10¹ >6.64 High POOA, 15 <1.0 × 10¹ >6.64 Low OA 20 <1.0 × 10¹ >6.64 25<1.0 × 10¹ >6.64 30 <1.0 × 10¹ >6.64 4 5 3.4 × 10⁵ 2.11 High H₂O₂, 101.6 × 10⁴ 3.44 High POOA, 15 1.9 × 10³ 4.36 Low OA 20 3.0 × 10¹ 6.17 25<1.0 × 10¹ >6.64 30 <1.0 × 10¹ >6.64 5 5 <1.0 × 10¹ >6.64 Low H₂O₂, 10<1.0 × 10¹ >6.64 Low POOA, 15 <1.0 × 10¹ >6.64 High OA 20 <1.0 ×10¹ >6.64 25 <1.0 × 10¹ >6.64 30 <1.0 × 10¹ >6.64 6 5 4.4 × 10⁶ 1.00High H₂O₂, 10 4.1 × 10⁵ 2.03 Low POOA, 15 7.7 × 10⁴ 2.76 High OA 20 5.3× 10⁴ 2.92 25 1.4 × 10⁴ 3.50 30 5.8 × 10³ 3.88 7 5 <1.0 × 10¹ >6.64 LowH₂O₂, 10 <1.0 × 10¹ >6.64 High POOA, 15 <1.0 × 10¹ >6.64 High OA 20 <1.0× 10¹ >6.64 25 <1.0 × 10¹ >6.64 30 <1.0 × 10¹ >6.64 8 5 1.2 × 10⁵ 2.56High H₂O₂, 10 2.0 × 10³ 4.34 High POOA, 15 4.0 × 10¹ 6.04 High OA 20<1.0 × 10¹ >6.64 25 <1.0 × 10¹ >6.64 30 <1.0 × 10¹ >6.64

A graphical representation of the reduction of B. cereus spores at 40°C. is presented in FIG. 1. The lower limit of detection for the testprocedure was 10 C.F.U./mL.

CONCLUSIONS:

Effect of H₂O₂:

The sporicidal activity of 150 ppm POAA at 40° C. against Bacilluscereus spores was most effective when in the presence of relatively lowconcentrations of H₂O₂ (≈36 ppm as in Formulas 1, 3, 5 and 7). ReducedB. cereus sporicidal efficacy was observed using POAA with the higherconcentrations of H₂O₂ (≈529 ppm as in Formulas 2, 4, 6 and 8).

Effects of Octanoic and Peroctanoic Acid:

The sporicidal activity of 150 ppm POAA at 40° C. against Bacilluscereus spores increased when the concentrations of octanoic orperoctanoic acid increased. This phenomenon was clearly evident informulas containing the high concentrations of H₂O₂ (formulas 2, 4, 6and 8).

On a weight basis, peroctanoic acid had a greater effect on thesporicidal efficacy of 150 ppm POAA against B. cereus than octanoicacid. An increase of 38 ppm POOA resulted in a greater log reduction ofB. cereus spores than an increase of 99 ppm octanoic acid. An additiveeffect was observed when POOA and octanoic acid were combined.

What is claimed:
 1. A method of sterilizing an article comprising: (a)mixing a first aqueous solution and a second aqueous solution to form asterilizing solution comprising at least 100 ppm peroxy acid and asufficient amount of phosphate buffering agent to provide thesterilizing solution with a pH of about 5 to about 7, wherein: (i) thefirst aqueous solution comprises a mixture of carboxylic acid, hydrogenperoxide, and water, and (ii) the second aqueous solution comprisesphosphate buffering agent and water; and (b) immersing the article inthe sterilizing solution for at least 5 minutes to sterilize thearticle; wherein the sterilizing solution contains no effective amountof organic corrosion inhibitor and has a weight ratio of peroxy acid tohydrogen peroxide of at least 2:1.
 2. A method according to claim 1,wherein the phosphate buffering agent comprises trisodium phosphate. 3.A method according to claim 1, wherein the sterilizing solutioncomprises a catalytic amount of a catalyst for peroxidation of thecarboxylic acid by the hydrogen peroxide.
 4. A method according to claim1, wherein the peroxy acid comprises a peroxy acid of at least one C1 toC12 carboxylic acid.
 5. A method according to claim 1, wherein theperoxy acid comprises a peroxy acid of at least one C1 to C8 carboxylicacid.
 6. A method according to claim 1, wherein the sterilizing solutioncomprises 1000 to 5000 ppm peroxy acid.
 7. A method according to claim1, wherein the peroxy acid comprises at least one of performic acid,peracetic acid, perpropionic acid, perbutanoic acid, perpentanoic acid,perhexanoic acid, perheptanoic acid, peroctanoic acid, pemonanoic acid,perundecanoic acid, and perdecanoic acid.
 8. A method according to claim1, wherein the first aqueous solution further comprises peroxycarboxylicacid.
 9. A method according to claim 1, wherein the article comprises apolymeric material.
 10. A method according to claim 9, wherein thepolymeric material comprises at least one of polypropylene,polytetrafluoroethylene, acrylonitrile/styrene/butadiene, polyolefin,polyvinyl chloride, polyvinyl butyrel, silicone resins, rubber, andpolyurethane.
 11. A method for sterilizing according to claim 1, whereinthe article comprises at least one of stainless steel, aluminum, brass,and copper.